PROJECT SUMMARY The assembly and release of HIV-1 from infected cells are essential steps in the viral replication cycle. HIV assembly is driven by the virally encoded Gag polyprotein. Bending of the plasma membrane into spherical buds, packing of the RNA genome (gRNA), and incorporation of the envelope glycoprotein (Env) are among the key events of assembly and budding. Release depends on the the host-encoded ESCRT proteins, which are recruited by Gag to the neck. Despite considerable progress in understanding structural and cellular mechanisms of HIV assembly, several fundamental questions remain unanswered. It is not understood how specific packing of gRNA is achieved, nor is it clear how Env incorporation into the viral membrane is directed. The molecular mechanism whereby the ESCRT proteins cut the membrane neck and release the virion also remains unknown. Gag consists of four domains: matrix (MA), capsid (CA), nucleocapsid (NC), and p6. MA binds to the plasma membrane lipid PI(4,5)P2 and is thought to have a key role in Env incorporation. CA forms a lattice that scaffolds membrane bending and positions the remaining domains. NC binds to RNA relatively non-specifically on its own, but when appropriately scaffolded on the membrane, specifically packages dimeric gRNA. Finally, p6 contains late domain motifs that recruit the ESCRT scission machinery. Our lab has taken the view that multiple low affinity interactions with Gag domains, which are brokered by the context of a deformable membrane, broker the highly specific events of Env incorporation, gRNA packaging, and ESCRT recruitment and membrane scission. We have pioneered the use of recombinant full-length myristoylated HIV- 1 Gag in conjunction with deformable giant unilamellar vesicles (GUVs). In previous work, we reconstituted the recruitment of the entire ESCRT cascade by Gag assemblies on GUVs, and we showed how in the context of the GUV membrane, NC acquired the ability to specifically bind the gRNA packaging signal in the background of a large excess of competitor RNA. We have recently adopted a new technology for studying HIV assembly and release using membrane nanotubes pulled from GUVs using an optical trap. This system allows us to set the curvature of the membrane to defined a value. By encapsulating materials inside the GUV, we can recreate the topology of membrane scission by ESCRTs. Using a system for photo-uncaging ATP within the GUV by single vesicle UV illumination, we have been able to optically trigger membrane scission, visualize it, and measure mechanical forces associated with it. Indeed, a major advantage of the nanotube technique is that the mechanical forces associated with each step in the process can be read out using the optical tweezers. Using these tools, we are in a unique position to resolve the mechanism of ESCRT-mediated membrane scission, to understand the coupling of Gag to the ESCRTs, to define how gRNA and Env are packaged, and to determine if gRNA, Env, and ESCRT interations impact one another.